Many diagnostic tests for a component of a biological fluid result in the formation of an oxidizing agent such as hydrogen peroxide as a product. There is need, therefore, for a quantitatively accurate method for the determination of such oxidizing agents. For example, a successful method for the determination of glucose involves the treatment of the biological fluid containing glucose with glucoseoxidase to yield, inter alia, hydrogen peroxide as a product. The amount of hydrogen peroxide formed is then determined to measure the glucose content. Further, in the determination of uric acid in a biological fluid by the enzymatic method, any uric acid present is converted by uricase into allantoin and hydrogen peroxide. Subsequent determination of the peroxide formed provides the uric acid content. Also, cholesterol content is determined, after prior cleavage of cholesterol esters, using cholesteroloxidase, from the hydrogen peroxide formed.
However, such compositions and methods to be useful for the determination of the oxidizing agent formed must meet certain criteria, i.e., they must be adaptable to use with small amounts of specimen, must be simple enough to be used effectively in a clinical situation and must be sufficiently economical for mass screening. In addition, the method must be readily adaptable to an automated sequential or continuous flow system in order that a great many samples may be processed rapidly and with the highest possible accuracy. Further the composition used must have sufficient stability on storage to be readily available in clinical laboratories. Finally, the color developed must have sufficient sensitivity, i.e., be of a high intensity and have a favorable absorption region, i.e., such as not to be interfered with by the absorption regions of the other materials present.
In the glucose, uric acid and cholesterol tests mentioned above, hydrogen peroxide which results from the enzymatic conversion is colorimetrically determined by an additional step utilizing peroxidase and a color-forming composition, i.e. a chromogen. The following illustrates the reaction EQU H.sub.2 O.sub.2 + Chromogen .sup.Peroxidase Color Complex + H.sub.2 O.
One chromogen which has been so used is o-dianisidine, [C.sub.6 H.sub.3 (OCH.sub.3)NH.sub.2 ].sub.2. However, the color complex formed using this compound not only has a slight sensitivity (low color intensity) but also has absorption peaks in an unfavorable region (420-460 nm).
Another chromogen, proposed by W. Gochman and J. M. Schmitz [in Clinical Chemistry, vol. 17, No. 12, pp. 1154-1159 (1971)] is a mixture of 3-methyl-2-benzothiazole-hydrazone hydrochloride and N,N-dimethylaniline. This chromogen produces a color of high intensity which has absorption peaks in a favorable region (590-600 nm). However, this mixture has very poor shelf stability--i.e., less than a day--and thus is not amenable to routine use in a clinical laboratory.
Use of the compositions of this invention provides a reliable, convenient method for determining the quantity of oxidizing agent which results from enzymatic treatment of biological fluids. The method, using the compositions of this invention, is adapable for use in an automated sequential or continuous flow system as well as manual systems. Further, the compositions and methods of this invention overcome many of the disadvantages of the prior art compositions and methods. The compositions of this invention have greater shelf and storage stability than the prior art compositions and when used according to the method of this invention result in a developed color which has desirable intensity and absorption characteristics.